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Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases, including Pierce's disease of grapevines. Disease manifestation by X. fastidiosa is associated with the expression of several factors, including the type IV pili that are required for twitching motility. We provide evidence that an operon, named Pil-Chp, with genes homologous to those found in chemotaxis systems, regulates twitching motility. Transposon insertion into the pilL gene of the operon resulted in loss of twitching motility (pilL is homologous to cheA genes encoding kinases). The X. fastidiosa mutant maintained the type IV pili, indicating that the disrupted pilL or downstream operon genes are involved in pili function, and not biogenesis. The mutated X. fastidiosa produced less biofilm than wild-type cells, indicating that the operon contributes to biofilm formation. Finally, in planta the mutant produced delayed and less severe disease, indicating that the Pil-Chp operon contributes to the virulence of X. fastidiosa, presumably through its role in twitching motility.

Xylella fastidiosa , a bacterium responsible for Pierce's disease in grapevines, possesses both type I and type IV pili at the same cell pole. Type IV pili facilitate twitching motility, and type I pili are involved in biofilm development. The adhesiveness of the bacteria and the roles of the two pili types in attachment to a glass substratum were evaluated using a microfluidic flow chamber in conjunction with pilus-defective mutants. The average adhesion force necessary to detach wild-type X. fastidiosa cells was 147 ± 11 pN. Mutant cells possessing only type I pili required a force of 204 ± 22 pN for removal, whereas cells possessing only type IV pili required 119 ± 8 pN to dislodge these cells. The experimental results demonstrate that microfluidic flow chambers are useful and convenient tools for assessing the drag forces necessary for detaching bacterial cells and that with specific pilus mutants, the role of the pilus type can be further assessed.

Xylella fastidiosa, an important phytopathogenic bacterium, causes serious plant diseases including Pierce's disease of grapevine. It is reported here that type I and type IV pili of X. fastidiosa play different roles in twitching motility, biofilm formation and cell-cell aggregation. Type I pili are particularly important for biofilm formation and aggregation, whereas type IV pili are essential for motility, and also function in biofilm formation. Thirty twitching-defective mutants were generated with an EZ : : TN transposome system, and several type-IV-pilus-associated genes were identified, including fimT, pilX, pilY1, pilO and pilR. Mutations in fimT, pilX, pilO or pilR resulted in a twitch-minus phenotype, whereas the pilY1 mutant was twitching reduced. A mutation in fimA resulted in a biofilm-defective and twitching-enhanced phenotype. A fimA/pilO double mutant was twitch minus, and produced almost no visible biofilm. Transmission electron microscopy revealed that the pili, when present, were localized to one pole of the cell. Both type I and type IV pili were present in the wild-type isolate and the pilY1 mutant, whereas only type I pili were present in the twitch-minus mutants. The fimA mutant produced no type I pili. The fimA/pilO double mutant produced neither type I nor type IV pili.

Fourteen Xylella fastidiosa isolates from grapevines exhibiting Pierce's disease symptoms in California, Texas, and South Carolina were examined for type IV pilus-mediated twitching motility, a phenotype previously observed in a Temecula isolate from California. All isolates except one from South Carolina (SC 19A97) exhibited colonies with a peripheral fringe on PW agar, a feature indicative of twitching motility; however, when individual cells of SC 19A97 were examined at higher magnifications twitching motility was observed. The presence and width of colony peripheral fringes were related to the amount of bovine serum albumin (BSA) present in the medium; no or low levels of BSA (0-1.8 g L(-1)) permitted development of the widest fringe, whereas higher levels (3.5-6.0 g L(-1)) severely limited, and in many instances prevented, peripheral fringe development. The growth rate of the wild-type Temecula isolate in PW broth with different concentrations of BSA was similar for all tested concentrations of BSA; however, growth was significantly reduced in medium without BSA.

It has been shown that conidia of Phyllosticta ampelicida require attachment to a substratum to initiate germination. Furthermore this attachment occurs only on hydrophobic surfaces. This study was initiated to ascertain the breadth of this phenomenon among other species of the genus Phyllosticta. We tested 23 isolates of Phyllosticta representing at least 14 named species. These isolates were collected from North America, Asia and Africa. For 22 of the 23 isolates tested spore attachment occurred at a rate of 60-100% on hydrophobic polystyrene but at 0-5% on hydrophilic polystyrene. The one exception to the preference for a hydrophobic substratum for attachment was an unnamed species of Phyllosticta from Rhus glauca that attached less than 10% on either surface. A similar response was observed when assaying germination and appressorium formation for 17 isolates. Germination and appressorium formation for these isolates proceeded on hydrophobic polystyrene but not on nutrient agar, which is hydrophilic. In five of the tested isolates germination was high on both hydrophobic polystyrene and hydrophilic nutrient media. The isolate from Rhus glauca did not germinate appreciably on either surface. Taken together these results suggest that the requirement for conidium contact/attachment to trigger germination is pervasive to the genus Phyllosticta.

Xylella fastidiosais a xylem-limited nonflagellated bacterium that causes economically important diseases of plants by developing biofilms that block xylem sap flow. How the bacterium is translocated downward in the host plant's vascular system against the direction of the transpiration stream has long been a puzzling phenomenon. Using microfabricated chambers designed to mimic some of the features of xylem vessels, we discovered thatX. fastidiosamigrates via type IV-pilus-mediated twitching motility at speeds up to 5 μm min−1against a rapidly flowing medium (20,000 μm min−1). Electron microscopy revealed that there are two length classes of pili, long type IV pili (1.0 to 5.8 μm) and short type I pili (0.4 to 1.0 μm). We further demonstrated that two knockout mutants (pilBandpilQmutants) that are deficient in type IV pili do not twitch and are inhibited from colonizing upstream vascular regions in planta. In addition, mutants with insertions inpilBorpilQ(possessing type I pili only) express enhanced biofilm formation, whereas a mutant with an insertion infimA(possessing only type IV pili) is biofilm deficient.

Two fluorophores, Solophenyl Flavine 7GFE 500 and Pontamine Fast Scarlet 4B, not heretofore reported upon are described as useful dyes of fungal cell walls, septa and bud scars examined microscopically. The dyes, depending on the filter sets used, yield fluorescently stained material generally in the blue to green and yellow to red wavelengths for Solophenyl Flavine 7GFE 500 and Pontamine Fast Scarlet 4B, respectively. They provide an excellent alternative to the more commonly used fluorophore, Calcofluor White M2R. The two fluorophores, in addition to being used at various spectral wavelengths from mercury arc sources, can be used with laser sources providing 488 nm and 543 nm line wavelengths, common to most scanning confocal microscopes. Unlike Calcofluor, Solophenyl Flavine 7GFE 500 and Pontamine Fast Scarlet 4B do not fade quickly when exposed to selected light wavelengths; however, like Calcofluors they are compatible with living fungal cells.

Colletotrichum graminicola, like many plant pathogenic fungi develop appressoria on germling apices, to facilitate penetration of their host. Induction of these structures occurs after contact with the host surface has been established by the germling. Surface contact and subsequent development of appressoria by germlings of C. graminicola was assessed using interference-reflection microscopy (IRM) and microfabricated pillared silicon substrata. Observations with IRM revealed that under low nutrient conditions, 90% of the germlings developed appressoria once they established 4.5 microm of continuous contact with the substratum. Substrata bearing pillars < or =5 microm in width supported < or =10% appressoria; however, as pillar width was increased the percentage of appressoria formed increased in a sigmoid fashion to a maximum of 80%. The percentage of appressoria produced experimentally on these surfaces was compared to data sets generated from a model designed to calculate the probability of appressorium development on similar pillar arrays at various germ tube contact lengths. These results indicate that germ tubes of C. graminicola require more than 4microm of continuous contact with a hydrophobic substratum for induction of appressoria.

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases including Pierce's disease of grapevines. X. fastidiosa is thought to induce disease by colonizing and clogging xylem vessels through the formation of cell aggregates and bacterial biofilms. Here we examine the role in X. fastidiosa virulence of an uncharacterized gene, PD1671, annotated as a two-component response regulator with potential GGDEF and EAL domains. GGDEF domains are found in c-di-GMP diguanylate cyclases while EAL domains are found in phosphodiesterases, and these domains are for c-di-GMP production and turnover, respectively. Functional analysis of the PD1671 gene revealed that it affected multiple X. fastidiosa virulence-related phenotypes. A Tn5 PD1671 mutant had a hypervirulent phenotype in grapevines presumably due to enhanced expression of gum genes leading to increased exopolysaccharide levels that resulted in elevated biofilm formation. Interestingly, the PD1671 mutant also had decreased motility in vitro but did not show a reduced distribution in grapevines following inoculation. Given these responses, the putative PD1671 protein may be a negative regulator of X. fastidiosa virulence.

Nanotechnology is having an enormous impact on many areas of science, including biology. However, its application to mainstream biology is limited by the complexity and expense of fabricating large numbers of silicon devices in nanofabrication facilities that are not readily available to all investigators. Biological experiments require a relatively large number of replicates. We have developed methods to use silicon devices as masters to mold replicas in polymers. Once the silicon master is fabricated, the method can be implemented in a standard biology laboratory. Polystyrene microdevices were fabricated for biological applications. Fabrication of such devices is described using hot embossing with and without vacuum, and by in situ polymerization from partially polymerized styrene monomers in polydimethylsiloxane molds made from silicon masters. Replication of the silicon master features into plastic using the polydimethylsiloxane intermediate mold is highly accurate for micrometer and sub-micrometer features. Preliminary biological experiments using polystyrene replicas have successfully demonstrated the potential of this fabrication method.

Pycnidiospores of Phyllosticta ampelicida germinate only after they become attached to a hydrophobic substratum. They do not readily attach or germinate on hydrophilic surfaces or on nutrient-rich media. The properties of the spore surface that function in attachment and signal mediation for germination were investigated in this study. Pycnidiospores were surrounded by a sheath that bound Concanavalin A (Con A)-TRITC and only weakly bound wheat germ agglutinin (WGA)-FITC. Galanthus nivalis lectin-FITC selectively bound primarily to the apical region of the sheath associated with the appendage. Con A, but not WGA or GNL, disrupted spore attachment. The Con A effect on spore attachment was significantly reduced when the lectin was pre-mixed with methyl-D-α-mannoside. The sheath as well as the associated appendage was removed with either Novozym 234 or crude β-glucuronidase. Such sheathless spores were initially adhesion incompetent but, by 40 min, the spores regained their ability to adhere to hydrophobic surfaces through the secretion of material that had an affinity toward colloidal gold. Non-treated pycnidiospores surrounded by sheath remained spatially separate; however, sheathless spores readily agglutinated. Addition of cations enhanced attachment to both hydrophobic and hydrophilic surfaces presumably through the masking of electronegative groups on the sheath or by reduction of the ordered water-shell surrounding the sheath. In particular, Ca 2+ induced both attachment and germination on normally non-inductive hydrophilic surfaces, suggesting an additional role for Ca 2+ in signal mediation for germination.

Calcium has been implicated in growth and appressorium formation of urediospore germlings of the bean rust fungus, Uromyces appendiculatus. Using ion microscopy, a mass spectrometry-based imaging technique, intracellular stores of calcium were analyzed by direct imaging of total calcium in frozen freeze-dried germlings. Calcium concentration was calculated by ratioing and spatially registering (40)Ca to (12)C signals. Intracellular distributions of total potassium, sodium, magnesium, and carbon were similarly imaged in the same germlings for a direct comparison of their localizations to total calcium. Calcium was remarkably heterogeneous with highest concentrations (2 to 10 mM) in the mid-region of the germling between the nuclei and the apex. A similar distribution of Ca(2+) (assessed using Fluo-3) was also noted sequestered in organelles in live germlings. Distributions of remaining elements (K, Na, Mg, and C) were mostly homogeneous throughout the cytoplasm and nuclei of the fungal cell. The K/Na ratio ranged from 17 to 31.

The existence of Ca2+-dependent protease II in crude extracts ofNeurospora crassa andUromyces appendiculatus was demonstrated by immunoblotting using specific antibodies. In both extracts two immunoreacting bands were observed. The molecular mass of the major band inN. crassa corresponded to 37 kDa, while that inU. appendiculatus was 43 kDa, similar to that previously reported forAllomyces arbuscula. Immunofluorescence of the enzyme was predominantly localized in the apical regions of germlings and growing hyphae, suggesting a functional role for the enzyme in hyphal growth.

Germlings of Uromyces appendiculatus respond to topographical stimuli and develop appressoria. During this period several genes are expressed, one of which is Inf 24. Germlings were microinjected with a sense and antisense fragment of the ORF of Inf 24. Sense ( Inf 24-S), 5′-ATG AGT GCT TCA TCG CAG CAG CAG-3′ fragment did not affect germling response to the topographical stimuli and the developed appressoria. Microinjection of antisense ( Inf 24-RC), 5′-CTG CTG CTG CGA TGA AGC ACT CAT-3′ blocked gene transcription and appressoria were rarely formed. Injection of Inf 24-RC into already formed appressoria did not inhibit continued development of subsequent infection structures, e.g. penetration peg, sub-stomatal vesicle.

Germination of Phyllosticta ampelicida pyc- nidiospores was followed from attachment through development of mature, highly melanized appresso- ria. Using time- lapsed video microscopy, it was de- termined that mature appressoria formed within 6 hours of attachment of the spores on hydrophobic substrata. Pycnidiospores germinated in the presence of tricyclazole did not produce pigmented appresso- ria indicating that the darkening agent is melanin, which in normal appressoria reached maximum lev- els within 6 hours following germination. Pycnidio- spores were binucleate. Prior to development of the septum separating the pycnidiospore and germ tube from the appressorium, the two nuclei divided mi- totically and three of the four resulting nuclei mi- grated into the appressorium. The fourth nucleus re- mained in the pycnidiospore where it degraded ul- timately. Mature appressoria formed penetration pegs capable of penetrating polycarbonate.

Phyllosticta ampelicida, the causal agent of black rot of grape, is one of the most damaging fungal pathogens in viticulture in North America. The interactions of the pathogen with host leaf tis- sues were studied to better understand the behavior

Germlings of the plant pathogenic fungusUromyces appendiculatus sense and respond to topographical signals of various substrata by undergoing a cell differentiation process that culminates in a structure termed an appressorium. In some cell systems, recognition and mediation of extracellular signals is via transmembrane glycoproteins known as integrins that often exhibit specific affinities to the tripeptide sequence Arg-Gly-Asp (RGD) found in several extracellular matrix components. Germlings grown on substrata inductive for appressorium formation in the presence of buffered synthetic peptides containing the amino acid sequence RGD, e.g., RGD, RGDS, GRGD, and GRGDGSPK (0.5–2.0 mM), were inhibited from developing appressoria. Two non-RGD peptides (GGGG and RGES) as well as two RGD peptides (GRGDS and RGDSPASSKP) did not inhibit appressorium formation. Germling growth was not significantly affected by any of the peptides. Furthermore, 0.5 μm diameter micropipettes that are normally inductive for appressorium formation when positioned between the germling apex and the substratum did not induce appressorium formation when coated with the RGD peptide. Silanized micropipettes left uncoated or coated with RGES were inductive for appressorium formation. Those observations lead to the hypothesis that an integrin-like protein may be involved in the process of signaling for initiation of appressorium formation inUromyces. An RGDSPC-affinity column was used to isolate proteins fromUromyces germlings with affinity to the RGD sequence. Elution with RGD or EDTA, but not with RGES, yielded at least 12 proteins of which one protein (95 kDa) expressed affinity on immunoblots to two different antibodies of β1-integrin; one to the carboxyl-terminus of a synthetic peptide of integrin from chicken, and the other from the amino terminus of integrin from human placenta.

Pycnidiospores of Phyllosticta ampelicida, the causal agent of black rot of grape, were found to germinate only on substrata on which they were firmly attached. Such surfaces were poorly wettable and had advancing contact angles (straight thetaa) formed by a water drop of80deg;, e.g., grape leaf, polystyrene, Teflon, polycarbonate, collodion, and glass treated with the silanes n-octadecyltrichlorosilane, dimethyldichlorosilane, or diphenyldichlorosilane. When pycnidiospores were deposited on more wettable surfaces they did not attach firmly and did not germinate. Such highly wettable surfaces had straight thetaa/= 40deg; and were represented by heat-treated glass, cellophane, nutrient- and water-agars, polystyrene treated with UV-irradiation or sulfuric acid, and glass silanized with n-2-aminoethyl-3-aminopropyltrimethoxysilane, n-(trimethoxysilylpropyl)ethylenediamine triacetic acid trisodium, or 3-aminopropyltriethoxysilane. Adhesion of pycnidiospores was assessed with and without a hydraulic shearing force. Pycnidiospore adhesion occurred over several minutes in distilled deionized water, unless it was first acidified, which decreased attachment time to0.03 s. Attachment of pycnidiospores treated with sodium azide, formaldehyde, or boiled in water for 10 min was similar to nontreated conidia. Possible mechanisms of adhesion of the conidia to surfaces include hydrophobic and ionic interactions.

Lamboy, J. S., Staples, R. C., and Hoch H. C. 1995. Superoxide dismutase: A differentiation protein expressed in Uromyces germlings during early appressorium development. Experimental Mycology 19, 284-296. Germlings of the bean rust fungus Uromyces appendiculatus detect penetration sites on the surface of the host leaf by thigmosensing topographical features. Within 2-4 min after the apex of a urediospore germ tube encounters the cuticular lip of a stomate, the germling ceases polarized growth and begins to swell over the aperture. The mechanism by which the cells detect topographical signals is not understood; however, previous experiments indicated that the initiation process does not involve de novo gene expression. In order to detect posttranslational modifications, the protein profiles of induced and noninduced germlings were compared at the earliest stages of appressorium formation, and a 21-kDa differentiation protein was identified by a shift in isoelectric point. The N-terminal amino acid sequence exhibited homology with superoxide dismutase (SOD), and antibodies to a synthetic peptide fragment of the respective sequence recognized cooper/zinc isozymes of SOD in electroblots of native gels. Electroelution of the active enzyme bands and separation by SDS-PAGE indicated that the 21-kDa protein is a component of a tetrameric 85-kDa SOD.

The presence and extent of the extracellular matrix surrounding pycnidiospore germlings and appressoria of Phyllosticta ampelicida was assessed using cryo-scanning and light microscopy. Conidia of ...

The sites alongUromyces appendiculatus germ tubes that are responsive to topographical induction for appressorium formation were determined using glass micropipettes. The germ tubes were perturbed with the micropipettes at different sites and durations. The most responsive region of the germ tubes for appressorium formation was within 0–10 μm from the cell apex where >90% of the perturbed germ tubes developed appressoria. Furthermore, only the cell surface in contact with the substratum was responsive. Appressoria could not be induced to form, under any conditions, by perturbing cell-substratum regions of the germ tubes more than 40 μm from the apex. Maximum appressorium formation occurred when the perturbing micropipette was left in place for >20 min.

Secreted proteins and other components of the Uromyces appendiculatus urediospore germling ex? tracellular matrix were fractionated by a scheme em- ploying a hydrophobic-silanized, fumed silica matrix. Two fractions of secreted proteins were obtained by this procedure. One fraction was adsorbed by hydro- phobic interactions from germination water filtrate. The second group of components, apparently less hy- drophobic in character and left behind in solution, was subsequently fractionated by anion-exchange chromatography. The total protein and glycoprotein profiles of several fractions were compared after so? dium dodecyl sulfate polyacrylamide gel electropho? resis. Batch extraction of putative adhesion proteins by silanized fumed silica matrices is proposed as a one- step procedure for extracellular matrix antigen prep? aration.

Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of bacterial wilt and canker of tomato, is the most destructive bacterial disease of tomato causing substantial economic losses in Israel, the U.S.A. and worldwide. The molecular strategies that allow Cmm, a Gram-positive bacterium, to develop a successful infection in tomato plants are largely unknown. The goal of the project was to elucidate the molecular interactions between Cmmand tomato. The first objective was to analyze gene expression profiles of susceptible tomato plants infected with pathogenic and endophytic Cmmstrains. Microarray analysis identified 122 genes that were differentially expressed during early stages of infection. Cmm activated typical basal defense responses in the host including induction of defense-related genes, production of scavenging of free oxygen radicals, enhanced protein turnover and hormone synthesis. Proteomic investigation of the Cmm-tomato interaction was performed with Multi-Dimensional Protein Identification Technology (MudPIT) and mass spectroscopy. A wide range of enzymes secreted by Cmm382, including cell-wall degrading enzymes and a large group of serine proteases from different families were identified in the xylem sap of infected tomato. Based on proteomic results, the expression pattern of selected bacterial virulence genes and plant defense genes were examined by qRT-PCR. Expression of the plasmid-borne cellulase (celA), serine protease (pat-1) and serine proteases residing on the chp/tomA pathogenicity island (chpCandppaA), were significantly induced within 96 hr after inoculation. Transcription of chromosomal genes involved in cell wall degradation (i.e., pelA1, celB, xysA and xysB) was also induced in early infection stages. The second objective was to identify by VIGS technology host genes affecting Cmm multiplication and appearance of disease symptoms in plant. VIGS screening showed that out of 160 tomato genes, which could be involved in defense-related signaling, suppression of 14 genes led to increase host susceptibility. Noteworthy are the genes Snakin-2 (inhibitor of Cmm growth) and extensin-like protein (ELP) involved in cell wall fortification. To further test the significance of Snakin -2 and ELP in resistance towards Cmm, transgenic tomato plants over-expressing the two genes were generated. These plants showed partial resistance to Cmm resulting in a significant delay of the wilt symptoms and reduction in size of canker lesion compared to control. Furthermore, colonization of the transgenic plants was significantly lower. The third objective was to assess the involvement of ethylene (ET), jasmonate (JA) and salicylic acid (SA) in Cmm infection. Microarray and proteomic studies showed the induction of enzymes involved in ET and JA biosynthesis. Cmm promoted ET production 8 days after inoculation and SIACO, a key enzyme of ET biosynthesis, was upregulated. Inoculation of the tomato mutants Never ripe (Nr) impaired in ET perception and transgenic plants with reduced ET synthesis significantly delayed wilt symptoms as compared to the wild-type plants. The retarded wilting in Nr plants was shown to be a specific effect of ET insensitivity and was not due to altered expression of defense related genes, reduced bacterial population or decrease in ethylene biosynthesis . In contrast, infection of various tomato mutants impaired in JA biosynthesis (e.g., def1, acx1) and JA insensitive mutant (jai1) yielded unequivocal results. The fourth objective was to determine the role of cell wall degrading enzymes produced by Cmm in xylem colonization and symptoms development. A significance increase (2 to 7 fold) in expression of cellulases (CelA, CelB), pectate lyases (PelA1, PelA2), polygalacturonase and xylanases (XylA, XylB) was detected by qRT-PCR and by proteomic analysis of the xylem sap. However, with the exception of CelA, whose inactivation led to reduced wilt symptoms, inactivation of any of the other cell wall degrading enzymes did not lead to reduced virulence. Results achieved emphasized the complexity involved in Cmm-tomato interactions. Nevertheless they provide the basis for additional research which will unravel the mechanism of Cmm pathogenicity and formulating disease control measures.

Terhune, B. T., and Hoch, A. C. 1993. Substrate hydrophobicity and adhesion of Uromyces urediospores and germlings. Experimental Mycology 17, 241-252. Adhesions of urediospores and urediospore germlings of Uromyces appendiculatus, the bean rust pathogen, to various substrata was evaluated with regard to surface wettability. A range of surface wettabilities, or conversely hydrophobicities, was obtained by coating glass or quartz substrates with various organosilanes. Adhesion of urediospores or germlings was evaluated after the spore or germling laden-silanized surfaces were washed. Both urediospores and germlings adhered most tenaciously to surfaces with wettability ratings less than 30. Such surfaces were polystyrene and glass treated with dimethyldichlorosilane, (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichorosilane, and diphenyldichlorosilane. The degree of germling contact to the various surfaces correlated closely with hydrophobicity and with the adhesion of germlings. Induction of appressoria on quartz substrates bearing inductive topographies (0.5-μm-deep grooves) was also closely associated with the degree of hydrophobicity.

Correa, A., Jr., and Hoch, H. C. 1993. Microinjection of urediospore germlings of Uromyces appendiculatus. Experimental Mycology 17, 253-273. Urediospore germlings of Uromyces appendiculatus were microinjected with a variety of materials to assess the applicability of microinjection methodologies for studying fungal cell growth and development. Until now, hyphal cells have not been successfully microinjected without first being pretreated with osmotica to lower the turgor pressures of the cells. We describe the development of a hydraulicly actuated system and protocols for successfully microinjecting Uromyces cells on a routine basis. Synthetic fluorocarbon and silicone fluids, various fluorophore-conjugated dextrans, rhodamine-conjugated phalloidin, rhodamine 123, cyclic AMP, and cytochalasin E were all microinjected into living urediospore germlings that grew or responded in ways consistent with expected pharmacological effects of the injected material. Germlings that were microinjected with synthetic fluids maintained the ability to sense and respond to inductive topographies for the formation of appressoria. Materials were injected at volumes of ≤0.5 to ≤400 fl. The results of this investigation indicate that fungal cells can be successfully microinjected with a variety of materials and, importantly, continue to function as normal cells.

Terhune, B. T., Bojko, R. J., and Hoch, H. C. 1993. Deformation of stomatal guard cell lips and microfabricated artificial topographies during appressorium formation by Uromyces. Experimental Mycology 17, 70-78. The inductive signal, stomatal guard cell lips or 0.5-μm-high plastic ridges, for appressorium formation in urediospore germlings of Uromyces appendiculatus was examined for signs of physical deformation during the course of appressorium development. The normally erect stomatal guard cell lips were usually observed prostrate at most stages of appressorium development; and, there were no persistent or significant indentations into the fungal cell that might have been caused by the topographical features. To further evaluate the events that occurred at the lip-appressorium interface in situ, polycarbonate and/or polystyrene ridges (0.5 μm high and 0.25 μm wide), mimicking stomatal guard cell lips of Phaseolus vulgaris, were cast on specifically microfabricated silicon templates. These artificial lips induced appressoria and became deformed approximately 30 min after initial contact by the germ tube apex as recorded and observed with time-lapsed video light microscopy. The collapsed nature of the ridges was further evaluated by both transmission and scanning electron microscopy. These results suggest that mechanical forces imposed by a combination of cell turgor pressure and adhesion of the appressorium to the substrate were responsible for deformation of the inductive topography.

Aims: Agrobacterium vitis is the causal agent of crown gall of grapevine. Surface motility (swarming), an important mechanism for bacterial colonization of new environments and a previously unknown behaviour of Ag. vitis, was demonstrated. Methods: Surface motility assays were performed on half-strength potato dextrose agar (Difco) containing 0·75% agar. To test for surfactant production, a drop-collapse test was used. Quorum-sensing (QS) negative and complemented mutants were tested for swarming activity. Results: Ninety-one Agrobacterium strains representing –Agrobacterium tumefaciens (17 strains), Agrobacterium rhizogenes (14 strains) and Ag. vitis (60 strains) were tested for swarming and production of surfactant. All Ag. vitis strains expressed a surface-related motility. In contrast, none of 17 strains of Ag. tumefaciens or 14 strains of Ag. rhizogenes exhibited this behaviour. Surface motility in Ag. vitis was associated with surfactant secretion; both of which are regulated by a QS system previously associated with induction of a hypersensitive response on tobacco and necrosis on grape. An aviR (belongs to luxR family) mutant was surface motility negative and did not produce surfactant. An avsI mutant (autoinducer synthase) was also surface motility negative and was complemented with an Ag. tumefaciens clone expressing avsI. Conclusions: Agrobacterium vitis is able to produce a characteristic swarming phenotype that is regulated by a complex QS system. Significance and Impact of the Study: Swarming activity is unique to Ag. vitis among Agrobacterium sp. and may be associated with the ability of the pathogen to colonize grapevines.

A mutation in the Xylella fastidiosa tonB1 gene resulted in loss of twitching motility and in significantly less biofilm formation as compared with a wild type. The altered motility and biofilm phenotypes were restored by complementation with a functional copy of the gene. The mutation affected virulence as measured by Pierce's disease symptoms on grapevines. The role of TonB1 in twitching and biofilm formation appears to be independent of the characteristic iron-uptake function of this protein. This is the first report demonstrating a functional role for a tonB homolog in X. fastidiosa.

Xylella fastidiosa is able to form biofilms within xylem vessels of many economically important crops. Vessel blockage is believed to be a major contributor to disease development caused by this bacterium. This report shows that Vitis riparia xylem sap increases growth rate and induces a characteristic biofilm architecture as compared with biofilms formed in PD2 and PW media. In addition, stable cultures could be maintained, frozen and reestablished in xylem sap. These findings are important as xylem sap provides a natural medium that facilitates the identification of virulence determinants of Pierce's disease.

Uromyces appendiculatus germlings attached to a plastic surface and differentiating in response to contact with inductive ridges formed appressoria most efficiently in the pH range 5.5–6.5. Formation of appressoria was inhibited by increased concentrations of Ca 2+ and K + .

Urediospore germlings of Uromyces appendiculatus sense topographical signals inherent in host stomata over which they develop appressoria. The site of signal recognition by the fungus is invariably within the apical 6 μm of the germling. The organization of the germling apex before and during initiation of appressorium formation was examined by both electron microscopy and laser scanning confocal microscopy. Most notable were changes in the distribution and organization of the microtubules and apical vesicles. Microtubules were oriented parallel to the inductive ridge, along which expansion of the germling apex occurred. In germlings that contacted artificial inductive topographies in vitro, the distribution of apical vesicles in germlings did not change within the first 3–4 min. After 6–8 min of contact, most apical vesicles became repositioned near the ridge. The apical cluster in developing appressoria was closely associated with that population of cytoplasmic microtubules that developed parallel to the inductive ridge. Coincident with growth of the germling apex over an inductive ridge was a physical deformation of the cell by the ridge, indenting both the cell wall and plasma membrane. Key words: bean rust, cytoskeleton, infection structure, Spitzenkörper, urediospore.

Appressorium development in Uromyces appendiculatus was studied to establish temporal and spatial parameters possibly important in signaling mechanisms in this fungus. It was determined that urediospore germling apices sensed inductive topographies, e.g., precisely shaped ridges 0.5 μm high by 2.0 μm wide, within 4 minutes after initial contact. The area in the germling that senses such topographies was located on the substrate side of the cell and within 6.0 μm of the apex. These conclusions were based on observed changes in the rate of germling growth and nuclear migration as well as the timing of apical tip swelling that began 4 minutes after contact. DNA synthesis during appressorium development occurred only after the first nuclear division and was probably in preparation for the second mitosis that normally occurs in the substomatal vesicle. Treatments with the DNA synthesis inhibitors hydroxyurea and actinomycin D reduced the DNA content of individual nuclei only after nuclear division. These results indicate that the nuclei in urediospore germlings were already in the G2 phase when germination began. Formation of a septum during appressorium development was initiated 45–50 minutes after contact of the germling with the ridge and was completed within 60 minutes after all of the cytoplasm had entered into the swollen apex.

Formation of appressoria inUromyces appendiculatus is triggered by external physical features of host stomata as well as artifical surfaces bearing inductive topographies. Microtubule and F-actin microfilament cytoskeletons were examined for their involvement in the process of appressorium formation in this fungus, using laser scanning confocal and electron microscopy. In germlings not stimulated to form appressoria the microtubule and microfilament cytoskeletons were organized as filaments mostly oriented parallel to the longitudinal axis of the cell. Following contact of the germling apex with an inductive topographical signal, e.g., 0.5 μm high ridge, the microtubules and F-actin filaments in the cell apex nearest the substrate appeared randomly oriented. Microtubules farther from the substrate remained oriented parallel to the longitudinal axis of the cell. In later stages of appressorium development, many cytoskeletal elements became oriented parallel to the inductive ridge, especially near the substrate. In regions farther from the substrate in these same cells, the microtubules and microfilaments were arranged in a reticulate pattern. Changes in the distribution and organization of the microtubule and F-actin microfilament cytoskeletons reflect a change in cell function following signal reception for appressorium. The reorientation of the cytoskeleton likely dictates the change in cell morphology.

Hyphae and germinating conidia of European hypovirulent (dsRNA-containing) Cryphonectria parasitica strain Ep-50 and virulent (dsRNA-free) strain Ep-67 (isogenic derivative from Ep-50), and hyphae only of European hypovirulent (dsRNA-containing) strains Ep-4 and Euro-7 were freeze-substituted and examined for the presence of virus-like particles (VLPs) using transmission electron microscopy. Spherical, membrane-bounded VLPs, measuring 50–90 nm in diameter, were located in hyphae and conidia of hypovirulent strain Ep-50, but not virulent strain Ep-67. Hyphae of hypovirulent strains Ep-4 and Euro-7 contained VLPs similar to those found in Ep-50. The VLPs occurred in aggregates surrounded by rough endoplasmic reticulum or more rarely, scattered throughout the cytoplasm; most possessed an electron-dense core. Results of Bernhard's regressive staining technique and lipid extraction cytochemistry suggested that the particles consisted of RNA surrounded by a lipid membrane. A unique Golgi body was associated with the formation of VLPs in hypovirulent strain Ep-50. The VLPs do not resemble typical fungal viruses and may be the end product of a defense response on the part of the fungal host designed to wall off foreign nucleic acid. Key words: Endothia parasitica, mycovirus, defense response, hypovirulence, viroid, fungal Golgi.

Xylella fastidiosa possesses both type I and type IV pili at the same cell pole. By use of a microfluidic device, the speed of twitching movement by wild-type cells on a glass surface against the flow direction of media was measured as 0.86 (standard error [SE], 0.04) μm min −1 . A type I pilus mutant ( fimA ) moved six times faster (4.85 [SE, 0.27] μm min −1 ) and a pilY1 mutant moved three times slower (0.28 [SE, 0.03] μm min −1 ) than wild-type cells. Type I pili slow the rate of movement, while the putative type IV pilus protein PilY1 is likely important for attachment to surfaces.

Conidia of Phyllosticta ampelicida germinate only after they have made contact with a substratum. Previous work has shown that external free calcium must be available to the spore for germination to be initiated. Transgenic strains of P. ampelicida expressing apo-aequorin, a calcium-sensitive luminescent protein, were developed to monitor cytoplasmic free Ca(2+) ([Ca(2+)]c). Transformants were verified by PCR and Southern hybridization. Apo-aequorin production was quantified for each of 21 transformants. The transformant that emitted the most light per unit of protein was found to contain 0.59 mg apo-aequorin/g total protein. To ascertain the feasibility of aequorin-based [Ca(2+)]c quantification, [Ca(2+)]c changes were measured in mycelia during various physiologically perturbing treatments: exposure to high concentrations of external Ca(2+), hypoosmotic shock, and mechanical perturbation. This is the first report of a plant pathogenic fungus for which aequorin-based Ca(2+) measurement protocols have been developed.

Fungal cells respond to a multitude of environmental conditions for reproduction, dispersal, attachment to a substratum, growth, and in the case of pathogenic species penetration into host tissues. For phytopathogenic fungi, a set of prerequisite conditions must be met before a successful parasitic relationship can be established. The fungal propagule, usually a spore, is dispersed to the host surface and is secured to that surface in a way that optimizes the likelihood of remaining there until germination and growth occur. Optimally, growth is toward the most appropriate site for entry into the host. The environmental conditions that surround the fungal cell and promote these cell responses vary, but include minimally the availability of moisture, light, nutrients, and physical characteristics of the substratum such as wettability and topography.

Phyllosticta ampelicida conidia germinate only after making contact with and attaching to a substratum. Previous studies suggested a role for Ca 2+ in this process. A Ca 2+ buffering system was used to control the external free Ca 2+ concentration. Both germination and appressorium formation were reduced or abolished with low Ca 2+ (less than or equal to nanomolar levels) but were nearly 100% at millimolar levels of Ca 2+ . Germination initiation required Ca 2+ within 10–25 min after the spore made contact with the substratum. Appressorium initiation required Ca 2+ 90–120 min following initial contact. Ca 2+ channel blockers nicardipine and lanthanum abated spore development. TMB-8, a blocker of internal Ca 2+ channels, reduced both developmental events. Gadolinium, a putative stretch-activated Ca 2+ channel blocker, abolished both developmental events at nanomolar levels. Calmodulin antagonists, compounds R-24751 and 48/80, abated spore development at micromolar levels. Together, these results suggest that Ca 2+ signaling is involved in both germination and appressorium formation in P. ampelicida pycnidiospores.

Bean leaf stomata provide a topographical signal that induces germlings of the phytopathogen Uromyces appendiculatus to develop specialized infection structures. Protoplasts from germ tubes of this fungus, when examined with patch-clamp electrodes, displayed the activities of a 600-picosiemen mechanosensitive ion channel. This channel passes a variety of cations, including Ca2+, and is blocked by Gd3+ at 50 micromolar. This channel could transduce the membrane stress induced by the leaf topography into an influx of ions, including Ca2+, that may trigger differentiation.

Leaf surfaces provide an important substrate for the growth of a wide variety of fungal organisms. Saprophytic fungi obtain water and nutrients from these surfaces and the leaf surface provides a physical environment suitable for growth and reproduction. For phytopathogenic fungi, the leaf surface represents a temporary, supportive environment, but a surface that must be breached before a successful pathogenic interaction can become established. Thus, the physical and chemical characteristics of the leaf surface play an important role in governing the success or failure of fungal growth on, and subsequently in, the leaf. Numerous reviews have been written about the growth of fungi in association with plant surfaces (e.g., Preece, 1976; Aist, 1981; Wynn, 1981; Staples and Macko, 1984; Hoch and Staples, 1987, 1991). This chapter focuses on features of the leaf surface as they influence the establishment and growth of various fungi. Attention will be given to the time and place of initial spore deposition on the leaf, to growth over the surface, and in the case of pathogenic fungi, to their penetration into the leaf. Since the authors’ research experience lies in the growth and development of rust fungi, considerable emphasis will be placed on examples involving these organisms.

I. Spore Attachment and Invasion.- 1. Adhesion of Fungi to the Plant Surface: Prerequisite for Pathogenesis.- 2. Signaling for Infection Structure Formation in Fungi.- 3. The Plant Cell Wall as a Barrier to Fungal Invasion.- 4. Rust Basidiospore Germlings and Disease Initiation.- 5. Attachment of Mycopathogens to Cuticle: The Initial Event of Mycoses in Arthropod Hosts.- 6. The Fate of Fungal Spores in the Insect Gut.- 7. Candida Blastospore Adhesion, Association, and Invasion of the Gastrointestinal Tract of Vertebrates.- 8. Infectious Propagules of Dermatophytes.- II. Fungal Spore Products and Pathogenesis.- 9. Melanin Biosynthesis: Prerequisite for Successful Invasion of the Plant Host by Appressoria of Colletotrichum and Pyricularia.- 10. The Plant Cuticle: A Barrier to Be Overcome by Fungal Plant Pathogens.- 11. Appearance of Pathogen-Related Proteins in Plant Hosts: Relationships between Compatible and Incompatible Interactions.- 12. The Role of Cuticle-Degrading Enzymes in Fungal Pathogenesis in Insects.- 13. Potential for Penetration of Passive Barriers to Fungal Invasion in Humans.- 14. Dihydroxynaphthalene (DHN) Melanin and Its Relationship with Virulence in the Early Stages of Phaeohyphomycosis.- III. Host Response to Early Fungal Invasion.- 15. Invasion of Plants by Powdery Mildew Fungi, and Cellular Mechanisms of Resistance.- 16. Induced Systemic Resistance in Plants.- 17. The Plant Membrane and Its Response to Disease.- 18. The Fungal Spore: Reservoir of Allergens.- 19. Conidia of Coccidioides immitis: Their Significance in Disease Initiation.- 20. Cell-Mediated Host Response to Fungal Aggression.- 21. Suppression of Phagocytic Cell Responses by Conidia and Conidial Products of Aspergillus fumigatus.- IV. Molecular Aspects of Disease Initiation.- 22. Molecular Approaches to the Analysis of Pathogenicity Genes from Fungi Causing Plant Disease.- 23. Current Status of the Molecular Basis of Candida Pathogenicity.- Taxonomic Index.

Introduction: The frontiers and challenges L. W. Jelinski, H. C. Hoch, and H. G. Craighead 1. High resolution lithographic techniques for semiconductor nanofabrication A. Forchel, P. Ils, R. Steffen, M. Bayer 2. Principles of materials etching R. Germann 3.The development and application of micromechanical devices in biosystems K. D. Wise 4. Microsensors and microactuators for biomedical applications M. Esashi 5. The use of micromachined structures for the manipulation of biological objects M. Washizu 6. Light addressable potentiometric sensor: applications to drug discovery H. M. McConnell 7. A surface plasmon resonance biosensor for characterization of biospecific interactions M. Malmqvist 8. Standard test targets for high resolution light microscopy R. Oldenbourg, S. Inoue, R. Tiberio, A. Stemmer, G. Mei, and M. Skvarla 9. Cell constituent analysis: single cell sensitivity P. J. Beyer, R. A. Lee, M. R. Wood, N. Winograd, A. G. Ewing 10. Scanning force microscopy of biological macromolecules: present and future C. Bustamante, D. A. Erie, G. Yang 11. Nanoscale structures engineered by molecular self-assembly of functionalized monolayers D. L. Allara 12. Biofunctionalized membranes on solid surfaces R. Tampe, C. Dietrich, S. Gritsch, G. Elender, L. Schmitt 13. Molecular assembly technology for biosensors M. Aizawa, K. Nishiguchi, M. Imamura, E. Kobatake, T. Haruyama, Y. Ikariyama 14. Self-organized orderedgrowth of III-V semiconductor quantum wires K. Ploog 15. Formation of a simple model brain on microfabricated electrode arrays A. Kawana 16. Cellular engineering: control ofcell-substrate iInteractions P. E. Hockberger, B. Lom, A. Soekarno, K. E. Healy 17. Micro-control of neuronal outgrowth H. M. Buettner 18. Microfabricated surfaces in signaling for cell growth and differentiation in fungi H. C. Hoch, R. J. Bojko, G. L. Comeau, D. A. Lilienfeld 19. Effects of surface topography of implant materials on cell behavior in vitro and in vivo D. M. Brunette 20. Cell and growth cone behavior on micropatterned surfaces P. Clark 21. Force generation by the microtubule-based motor protein kinesin F. Gittes, E. Meyhofer, S. Baek, D. Coy, B. Mickey, J. Howard 22. Contemporary problems in biology: contractile materials G. H. Pollack 23. Technology needs for the human genome project David T. Burke Index.

The formation of urediniospore appressoria in response to micro-fabricated topographies was observed for 27 species of rust fungi representing eight genera. Nine species, including Uromyces appendiculatus, U. vignae, Melampsora medusae, Puccinia antirrhini, P. calcitrapae, P. carduorum, P. melanocephala, P. substriata, and one isolate of P. recondita, developed appressoria on ridges within an optimal height range of approximately 0.4-0.8 μm. Appressorium formation was considerably reduced on ridge heights above and below this range. A broader range of ridge heights was observed to be inductive for appressorium formation by P. polysora, P. menthae, P. hieracii, P. sorghi, P. arachidis, and Physopella zeae (...)

Germlings ofUromyces appendiculatus induced by exposure to 28.5°C heat for 1.5 h developed infection structures at nearly the same rate as those induced thigmotropically. Typically, the microtubules began to rearrange within 10 minutes of the start of heat treatment; nuclear migration followed. Mitosis started after 36 minutes. Appressorium development was accompanied by the appearance of six heat shock proteins, but not by the thigmotropic-specific proteins always observed during contact-induced development.